The laboratory is interested in understanding the clathrin-independent endocytosis pathway that is associated with Arf6. We have shown that this pathway is responsible for internalizing plasma membrane (PM) proteins that lack sequences that allow recognition by the clathrin and adaptor protein machinery. Among the proteins that enter cells via this mode of endocytosis are the major histocompatibility complex Class I protein (MHCI) and the lipid anchored protein CD59. After endocytosis, MHCI and CD59 in vesicles are delivered to and fuse with endosomes containing cargo proteins from the clathrin-dependent endocytosis pathway such as transferrin receptor. From there, MHCI and CD59 can proceed on to late endosomal compartments where they are degraded or they can be recycled back out to the PM via unique tubular recycling endosomes. In HeLa cells these recycling endosomes contain only cargo that had entered via clathrin-independent endocytosis and their return to the PM is dependent upon the activity of Arf6 and several other regulators including Rab11 and 22. [unreadable] [unreadable] We had previously shown that in addition to Arf6, Rac, a Rho GTP-binding protein that alters PM actin cytoskeleton, was associated with the clathrin-independent endosomal membranes and upon its activation resulted in PM ruffling and macropinocytosis, a stimulated form of clathrin-independent endocytosis. Recently we examined whether other signaling molecules (in addition to Arf6 and Rac) could be associated with this membrane system. We found that H-Ras is associated with these membranes and colocalizes with MHCI but then upon EGF treatment H-Ras is activated, resulting in PM ruffling and induction of macropinocytosis (Porat-Shliom et al 2008). Since H-Ras activation is known to result in the activation of a phosphotidylinositol 3-kinase, we examined the changes in phosphoinositide composition during the process of macropinocytosis. In cells expressing constitutively active mutant of H-Ras incoming macropinosomes initially contained phosphatidylinositol 4,5-bisphosphate (PIP2) and phosphatidylinositol 3,4,5-trisphosphate (PIP3). During maturation of the macropinosome, PIP2 was lost first, followed by the recruitment of Rab5 and then loss of PIP3. These sequential changes in phosphoinositide content were also seen in macropinosomes formed by activation of Arf6. The macropinosomes that form in response to activation of Arf6 or Ras do however recruit distinct effectors, for example Ras recruits Erk and Akt.[unreadable] [unreadable] In another project, we have isolated clathrin-independent endosomes in an attempt to identify machinery and additional cargo proteins associated with this pathway. Mass spectroscopy was used to identify new candidate proteins. We have validated 7 new cargo proteins that enter cells and traffic along this clathrin-independent pathway. The new proteins are: CD44, CD55, CD98, CD147, Lat1, ICAM1 and the non-insulin stimulated glucose transporter Glut1. We are currently examining in detail their pattern of endocytosis and trafficking within cells.